Collagen zymography as a sensitive and specific technique for the determination of subpicogram levels of interstitial collagenase.
We describe the use of casting native collagen type I in SDS-polyacrylamide gel (collagen zymography) for the determination of interstitial collagenase. As with gelatin, the incorporation of collagen in the gels reduced protein migration and the need for making corrections for an accurate Mr evaluation. This method proved to be very sensitive: 0.1 pg of APMA-activated procollagenase could be detected, and specific levels of active gelatinase or stromelysin lower than 5 ng were inactive under our experimental conditions. It was used to demonstrate the increased expression of collagenase following treatment of human gingival fibroblasts with interleukin-1 beta; the amounts of enzyme quantified by either collagen zymography or immunodot blot assay are comparable.
Gogly
B
B
Laboratory of Biology and Physiopathology Craniofacial, UFR Odontology, University René Descartes Paris V, Montrouge, France.
Groult
N
N
Hornebeck
W
W
Godeau
G
G
Pellat
B
B
D000328:Adult; D002478:Cells, Cultured; D003094:Collagen; D017364:Collagenases / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005110:Extracellular Space / Q000201:enzymology*; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D005881:Gingiva / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D015151:Immunoblotting; D007375:Interleukin-1 / Q000494:pharmacology; D008297:Male; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate
D007375:Interleukin-1; D012967:Sodium Dodecyl Sulfate; D003094:Collagen; D017364:Collagenases